Cytolytic peptides or cytolysins previously have been used to release active agents or “payload” from liposomes or cells. The mode of action for such peptides involves perturbation of the liposome or cellular membrane. These peptides include toxins from insects, fish, antibiotic peptides and synthetic peptides such as melittin, alamethicin, gramicidin, magainin and pardaxin, GALA, KALA, hemagglutinin subunit HA-2. Natural potent cytolytic peptides are found widely from insects to mammals, particularly as antimicrobial peptides or defensins, where they are involved in innate defense at mucosal membranes and as cytolysins in lymphocytes. In order to target and localize the cytolytic action of such peptides, specific steps (e.g. activating synthesis, release from lysosomes, and cleavage of propeptides) are required. The biological delivery activity of such peptides is tightly controlled. Cytolysin activity is cloaked by sophisticated mechanisms.
The acceptance of diagnostic methods involving the use of cytolysins has been hindered by their inferior sensitivity and non specific variable background, compared to alternative methods. Liposomes have, previously, been utilized in homogenous assays using complement-mediated lysis (Anal. Biochem. 1 18 (198 1) 286-293). However, such assays are considered unreliable as they involve many labile components, any one of which may become inactivated, thereby eliminating payload release. Preservation of the stability and water solubility of the cytolytic peptides remain obstacles to their use in diagnostic applications.
For a reliable assay, the release of detectable marker molecules should only occur in response to an external trigger and any leakage of marker molecules should be minimal for example, approaching zero, or at least remain constant over the assay period. Consequently, in such assays there is always a danger of background signal or interference caused by the progressive release of marker molecules. There is a need for better control over the activity of cytolysins for a variety of diagnostic assays.